1. INTRODUCTION 2. REACTIVE OXYGEN SPECIES 3. FREE RADICALS 4. ROLE OF R.O.S. 5. EFFECTS OF R.O.S. 6. OXIDATIVE DNA DAMAGE 7. SOURCES OF R.O.S. 8. R.O.S. & BODY'S DEFENSE SYSTEMS 9. MAPK PATHWAYS & DETOXIFICATION OF R.O.S. 10. DEFENSE MECHANISM 11. NRF2 ACTIONS 12. PROTEIN STRUCTURE OF NRF2 & KEAP1 13. ROLE OF NRF2 IN DEFENSE MECHANISMS

1. NRF2 RECEPTOR TRANSCRIPTION
FACTORS
PRIYANSHA SINGH (14/ 226)
M.S. (Pharm)- PHARMACOLOGY & TOXICOLOGY
NIPER GUWAHATI
educationpharmaiq@gmail.com

2. NRF2 : AN IMPORTANT TRANSCRIPTIONFACTOR
• Nuclear factor erythroid 2-related factor 2 (Nrf2) has been identified as a main
transcription factor that maintain cellular homeostasis through balancing of
signaling.
• Nrf2 signaling plays a crucial role in the cellular response to oxidative insult and
prevents damage to cellular components sensitive to redox changes.

3. REACTIVE OXYGEN SPECIES
• Reactive oxygen species (ROS) are chemically unstable free radicals and reactive
molecules containing molecular oxygen.
• Reactive oxygen species are generated as a by-product of many aerobic
metabolism and physiological processes.
• ROS includes :-
Free radicals: Superoxide anion (O2 .- ), Hydroxyl radical ( .OH), Lipid radicals,
Oxidizing agents: Hydrogen peroxide (H2O2 ), Peroxynitrite (ONOO− ), and
Hypochlorous acid (HOCl)

4. FREE RADICALS
• A free radical can be defined as any molecular species capable of independent
existence that contains an unpaired electron in an atomic orbital.
• Many radicals are unstable and highly reactive.
• They can either donate an electron to or accept an electron from other
molecules, therefore behaving as oxidants or reductants.

5. FREE RADICALS
• The most important oxygen-containing free radicals in many disease states are
hydroxyl radical, superoxide anion radical, hydrogen peroxide, oxygen singlet,
hypochlorite, nitric oxide radical, and Peroxynitrite radical.
• These are highly reactive species, capable in the nucleus, and in the membranes
of cells of damaging biologically relevant molecules such as DNA, proteins,
carbohydrates, and lipids

6. FREE RADICALS

7. REACTIVE OXYGEN SPECIES
• Reactive oxygen species (ROS) can be released by xanthine oxidase, NADPH oxidase,
cyclooxygenase, Lipoxygenase, mitochondrial respiration, cytochrome P450, or due to
the uncoupling of nitric oxide synthase (NOS) in vascular cells

8. REACTIVE OXYGEN SPECIES
• Superoxide dismutase (SOD), an endogenous enzyme catalyzes the superoxide free radical
(O2 .-) to hydrogen peroxide (H2O2) which can generate highly reactive hydroxyl radical
(.OH) by intermingling with transition metal ions (such as Fe and Cu), and extremely
reactive Hypochlorous acid (HOCl) through myeloperoxidase (MPO) enzyme.
• Moreover, nitric oxide (NO) reacts with superoxide radical (produced by NADPH oxidase,
xanthine oxidase and the mitochondrial respiration) generate an excessive amount of
Peroxynitrite (ONOO− ), a reactive nitrogen species which accelerates the structural
damage and causing further ROS production.

9. REACTIVE OXYGEN SPECIES
• Excessive production of ROS and the altered equilibrium between ROS and
endogenous antioxidant enzymes induces oxidative stress in the heart as well as in
the body.
• The excess generation of ROS targets cellular lipid, protein and DNA which
ultimately leads to tissue injury. Moreover, oxidative stress plays a crucial role in the
pathophysiology of cardiovascular and other diseases.
• Intriguingly, ROS acts as important intracellular/intercellular secondary messengers
to regulate many downstream signaling molecules including protein tyrosine
kinases, protein tyrosine phosphatases, transcription factors, mitogen-activated
protein kinases (MAPKs) and ion channels.

10. ROLES OF R.O.S

12. OXIDATIVE DNA DAMAGE

13. EFFECTS OF R.O.S. ON ORGANS

14. OXIDATIVE STRESS AND SOURCES OF R.O.S.
• A pathological condition in which production of ROS exceed the capacity of the
antioxidant system.
• ROS are short lived, chemically unstable, reactive molecules containing oxygen i.e.
peroxide, superoxide, hydroxyl radical and singlet oxygen.

15. SOURCES OF R.O.S.

16. R.O.S. AND BODY’S DEFENSE SYSTEM
• However, up to a certain limit, ROS are neutralized by
(a) endogenous antioxidant enzymes such as
o glutathione peroxidase (GPX),
o superoxide dismutase (SOD),
o glutathione S-transferase
o catalase

17. MAPK PATHWAYS & DETOXIFICATION OF R.O.S.

18. ANTIOXIDANT DEFENCE MECHANISM
• Inhibit excess ROS formation, capture radicals, maintain redox homeostasis and correct
mechanism of destroyed biomolecules
• Maintained through Antioxidant Responsive Element sequence
• Controlled by nuclear transcription factors Nrf2, AP-1, NF-kB, p53
• ANTIOXIDANT ENZYMES
❑Superoxide dismutase(SOD)
❑Catalase
❑Glutathione peroxidase(GPx)

19. THE ORIGIN AND FUNCTION OF NRF2
• Nrf2 is a leucine zipper/CNC protein, a polypeptide with a molecular weight of 66kDa.
• It is widely expressed in organs with hyperoxia consumption, such as the muscle, heart, vasculature,
liver, kidney, brain, lung, skin, and digestive tract.
• Under normal conditions, Nrf2 remains in the cytosol at a low concentration.
• Under stressful conditions, Nrf2 translocates into the nucleus and serves as a transcription factor to
to maintain cellular redox homeostasis.
• Nrf2 plays an important role in cellular resistance to oxidative stress and exogenous toxic substances,
and it is closely linked to inflammatory reactions, respiratory system diseases, cardiovascular diseases,
and malignant tumors.

20. NRF2 MEDIATED ANTI-OXIDATIVE RESPONSE
• Nrf2: is a redox-sensitive, Cap'n’Collar basic leucine zipper transcription factor that binds to
ARE to activate transcription. It contains seven functional domains Neh-1 (Nrf2-ECH
homology-1) to Neh-7.
• Keap1: Kelch-like ECH-associated protein 1 (Keap 1) interacts with Nrf2 in a redox sensitive
manner and is retained in the cytoplasm by associated with Keap1.

21. NRF2 & KEAP1 COMPLEX

22. NRF2 MEDIATED ANTI-OXIDATIVE RESPONSE

23. THE PROTEIN STRUCTURAL DOMAIN OF NRF2
• Nrf2 is a basic leucine zipper (bZIP) transcription factor belonging to the Cap 'n' Collar (CNC)
family.
• Studies have confirmed that Nrf2 is a polypeptide that contains 589 amino acid residues and
six domains, Neh1–Neh6 which are highly conserved among different species.
• Neh1 contains a bZIP motif through which Nrf2 interacts with Mafs and forms heterodimers
with DNA sequences.
• Neh2 mediates the formation of heterodimers of Nrf2 and Keap1, the latter of which is the
natural inhibitor of Nrf2 in the cytoplasm.
• Neh2 contains two motifs that combine with Keap1, an ETGE motif with strong affinity and a
DLG motif with weak affinity.

24. THE PROTEIN STRUCTURAL DOMAIN OF NRF2
• Neh2 also has a hydrophilic domain that is rich in lysine residues and is essential for Keap1-
dependent ubiquitin-mediated degradation of Nrf2.
• The Neh3 domain is located at the carboxyl terminus of Nrf2. The Neh4 and Neh5 domains
trigger the transcription of downstream ARE-dependent genes.
• The Neh6 domain is involved in non-Keap1-dependent regulation and degradation of Nrf2.
Because of the remarkable effects of Nrf2 on cell growth and apoptosis, DNA repair, inflammatory
responses, and redox conditions, there is widespread interest in defining the factors and
mechanisms that regulate its biological functions under physiological and pathological
conditions.
• The discovery that Keap1 is the key negative regulator of Nrf2 represents an important milestone
and the culmination of more than a decade of study and investigation.

25. THE PROTEIN STRUCTURAL DOMAINOF KEAP1
• Under physiological conditions, Nrf2 is bound to its inhibitory protein, Keap1, and
anchored to the actin cytoskeleton, which limits its transcriptional activity in the
nucleus.
• Keap1 is a polypeptide composed of 624 amino acid residues and five domains: NTR
(N-terminus), BTB/POZ, IVR, DGR, and CTR (C-terminus).
• The DGR domain contains six repetitive double-stranded glycine (Gly) sequences,
the binding sites of Nrf2

26. THE PROTEIN STRUCTURAL DOMAINOF KEAP1
• Keap1 contains two protein interaction motifs, BTB and Kelch, which are separated
by the IVR domain (Fig. 1A and B). The BTB/POZ domain contributes to the
formation of Keap1 homodimers, which are associated with Cul3/Rbx1–E3 ubiquitin
ligase.
• Ubiquitin ligase, which is also known as E3 ubiquitin ligase, connects ubiquitin
molecules to the lysine residues of proteins. Typically, ubiquitin ligase forms many
ubiquitin chains and is degraded by the 20S catalytic subunit of the proteasome

28. NRF2 MEDIATE ANTIOXIDANT RESPONSE

29. NRF2 MEDIATED ANTI-OXIDATIVE RESPONSE
• Under non-stressed conditions, cytosolic Nrf2 is suppressed through poly-
ubiquitination and proteasomal degradation by kelch-like ECH-associated protein-1
(Keap1) with an adaptor Cul3 (cullin-3)/Rbx1 (ring-box protein 1)- based E3
ubiquitin ligase complex.
• During oxidative stress, Nrf2 dissociates from Keap1 through modification in the
cysteine thiols group of Keap1 or via phosphorylation with members of mitogen-
activated protein kinases (MAPKs) such as extracellular signal regulated kinase (ERK),
p38 and c-June N terminal-kinase (JNK), phosphatidylinositide 3-kinases (PI3K),
PKR-like endoplasmic reticulum kinase (PERK) and protein kinase C (PKC).

30. NRF2 MEDIATEDANTI-OXIDATIVE RESPONSE
• These conformational changes prevent Nrf2 ubiquitination and proteasomal
degradation leading to nuclear translocation of Nrf2.
• In nucleus, Nrf2 heterodimerizes with small Maf proteins (MafG, MafF and MafK)
and enhances its binding to antioxidant response element (ARE) to activate the
transcription of Nrf2-dependent genes like
• heme oxygenase-1 (HO-1),
• NADPH quinine oxidoreductase 1 (NQO1),
• aldo-keto reductase (AKR),
• peroxiredoxin 1 (PRXD 1),
• glutathione-s-transferase (GST),
• glutathione peroxidase-1,2 (GPX-1,2),
• γ-glutamyl cysteine ligase-catalytic (γ-GCLC),
• γ-glutamyl cysteine ligase-modulatory (γ-GCLM) and
• superoxide dismutase (SOD).

31. NRF2 MEDIATE ANTIOXIDANT RESPONSE
• It subsequently combines with AREs to trigger the transcription of more than 200
endogenous protective genes, including
(i) Antioxidant genes,
(ii) Phase II detoxification enzyme genes,
(iii) Molecular chaperones, and
(iv) Anti-inflammatory co-stimulating genes.
• Furthermore, transcription of Nrf2 blocks the up-regulation of tumour necrosis
factor-α (TNF-α), interleukin-6 (IL-6), interleukin-17 (IL-17), interleukin-1β (IL-1β),
monocyte chemo-attractant protein-1(MCP-1), macrophage inflammatory protein-2
(MIP-2), and inhibits the promoter activity of vascular cell adhesion molecule-1
(VCAM-1)

32. NRF2 MEDIATED ANTI-OXIDATIVE RESPONSE

33. NRF2/ ARE SIGNALLINGAXIS

34. NRF2 & TARGET DISEASES

35. NRF2 & CARDIOVASCULAR DISEASE

36. NRF2 & KIDNEY DISEASES

37. NRF2 & BRAIN DISEASES